European Journal of Taxonomy 398: 1–18 ISSN 2118-9773 https://doi.org/10.5852/ejt.2018.398 www.europeanjournaloftaxonomy.eu 2018 · Atherton S. & Jondelius U. This work is licensed under a Creative Commons Attribution 3.0 License. Research article urn:lsid:zoobank.org:pub:58C075B0-7409-41B7-A6F4-900A5A6BFECE Microstomum (Platyhelminthes, Macrostomorpha, Microstomidae) from the Swedish west coast: two new species and a population description Sarah ATHERTON 1,* & Ulf JONDELIUS 2 1,2 Naturhistoriska riksmuseet, Box 50007, 104 05, Stockholm, Sweden. * Corresponding author: [email protected] 2 Email: [email protected] 1 urn:lsid:zoobank.org:author:1F597997-CD78-4F36-A82B-977B14DCAA6C 2 urn:lsid:zoobank.org:author:7F116C0B-A518-45D6-B62D-0C3B459D5F70 Abstract. Two new species of marine Platyhelminthes, Microstomum laurae sp. nov. and Microstomum edmondi sp. nov. (Macrostomida: Microstomidae) are described from the west coast of Sweden. Microstomum laurae sp. nov. is distinguished by the following combination of characters: rounded anterior and posterior ends; presence of approximately 20 adhesive papillae on the posterior rim; paired lateral red eyespots located level with the brain; preoral gut extending anterior to brain and very small sensory pits. Microstomum edmondi sp. nov. is a protandrous hermaphrodite with a single ovary, single testis and male copulatory organ with stylet. It is characterized by a conical pointed anterior end, a blunt posterior end with numerous adhesive papillae along the rim, and large ciliary pits. The stylet is shaped as a narrow funnel with a short, arched tip. In addition, the first records of fully mature specimens of Microstomum rubromaculatum von Graff, 1882 from Fiskebäckskil and a phylogenetic analysis of Microstomum Schmidt, 1848 based on the mitochondrial cytochrome oxidase I (COI) gene are presented. Keywords. Macrostomorpha, Macrostomida, flatworm, meiofauna, turbellaria. Atherton S. & Jondelius U. 2018. Microstomum (Platyhelminthes, Macrostomorpha, Microstomidae) from the Swedish west coast: two new species and a population description. European Journal of Taxonomy 398: 1–18. https://doi.org/10.5852/ejt.2018.398 Introduction Macrostomorpha Doe, 1986 is a group of free-living flatworms that may be found in aquatic and semi- aquatic habitats all over the world (Rieger 2001). Their relatively small size, typically only 1–2 mm (although animals as large as 5 mm have been recorded) and paucity of distinguishing morphological characters makes taxonomic identification within the group difficult (Janssenet al. 2015). Species identification of Macrostomorpha is largely dependent on the morphology of the reproductive system, and identification of species that predominately reproduce asexually, such as most species of Microstomum Schmidt, 1848, is particularly challenging. It is unsurprising, then, that while limited 1 European Journal of Taxonomy 398: 1–18 (2018) attention has been paid to species diversity within Macrostomorpha (e.g., Luo et al. 2011; Adami et al. 2012; Schockaert 2014; Sun et al. 2014; Janssen et al. 2015; Fang et al. 2016), current taxonomic research within the genus Microstomum is particularly scant (but see Rogozin 2015). Species of Microstomum primarily reproduce through asexual fission, but their life cycle may also include short periods of sexual reproduction (Bauchhenss 1971; Heitkamp 1982). During that time, individual zooids will develop both male and female sexual structures, including for the male complex: single or bilateral testes, vas deferentia, a male copulatory apparatus with a seminal vesicle, stylet and antrum masculinum, and a male gonopore; and for the female complex: a single ovary, female antrum and female gonopore. Microstomum sexual maturity may be present for as little as two weeks in a year (Bauchhenss 1971; Faubel 1974) and therefore descriptions of the reproductive organs exist for only 15 of the 31 currently accepted nominal species. Several members of Microstomum were encountered while investigating macrostomorph diversity in western Sweden. Herein, two new species of marine Microstomum are described, and a population of Microstomum rubromaculatum von Graff, 1882 from Fiskebäckskil is redescribed based upon the first records of fully sexual specimens. Additionally, we present a hypothesis regarding the phylogenetic position of the three species based on the mitochondrial cytochrome oxidase I (COI) gene and make COI barcode sequences available. Material and methods Sediments and aquatic vegetation were collected by hand from sites around the Sven Lovén Centre Kristineberg in Fiskebäckskil, Sweden on 17–20 Aug. 2015 and the Sven Lovén Centre Tjärnö in Strömstad, Sweden on 17–19 Jun. 2016. Exact dates and coordinates are listed in Table 1. Samples were transported back to the laboratory and marine flatworms were extracted within 48 hours following a standard MgCl2 anesthetization-decantation technique (Martens 1984). Animals were manually isolated under a Nikon SMZ 1500 stereo microscope, transferred to a glass slide and identified using a Nikon Eclipse 80i compound microscope equipped with DIC (differential interference contrast). Light micrographs and digital videos were captured with a Cannon EOS 5D Mark III digital camera. Measurements were taken with an ocular micrometer. Following documentation, individual specimens were fixed in 95% ethanol and transported to the Naturhistoriska riksmuseet in Stockholm for DNA extraction and analysis. DNA was extracted from whole animals using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. Amplification was performed via PCR using 0.2 ml PuReTaq Ready-To-Go PCR beads (GE Healthcare). A ~700 base-pair region of the COI gene was targeted using Mac_COIF+Mac_COIR (Janssen et al. 2015) or new (Mic_COIF 5’-GTT TGA GGA GGT TTG ATA GGC-3’; Mic_COIR 5’-ATC ACC CCC CTC CGT AGG AT-3’) PCR and sequencing primers, and amplified with the following program: 5 minutes hotstart at 94°C; followed by 40 cycles of 30 seconds at 94°C, 90 seconds at 50°C, and 60 seconds at 72°C; and a final extension time of 10 minutes at 72°C. Products were viewed on a 0.8% agarose gel, purified using ExoSAP-IT enzymes (Exonuclease and Shrimp Alkaline Phosphatase, GE Healthcare), and sent to Macrogen (Macrogen Europe, Netherlands) for commercial sequencing. Thirty-five COI sequences ofMicrostomum and seven outgroup sequences were used in the phylogenetic analysis (Table 1). The dataset included all COI sequences of Microstomum publically available in GenBank along with new sequences of the three species presented here. Outgroups were selected based on their position in the phylogenetic hypothesis of Janssen et al. (2015). Sequence assembly was performed in MEGA v. 6.06 (Darriba et al. 2012) and trace files were manually edited. Sequences were aligned as amino acids using the standard flatworm mitochondrial genetic code 2 ATHERTON S. & JONDELIUS U., Microstomum from West Sweden Table 1. List of specimens used in this study. The table lists specimens used in this study along with corresponding collection location, coordinates and date, as well as GenBank accession number, where available. * from Janssen et al. 2015; ** from Telford et al. 2000 Species GenBank # Location Coordinates Date Munkedal, 58°27′3″ N, MF185700-3 15 Aug. 2015 Sweden 11°41′10″ E Microstomum edmondi Fiskebäckskil, 58°14′52″ N, MF185704-11 17 Aug. 2015 Sweden 11°27′05″ E 58°52′41″ N, MF185712 Saltö, Sweden 19 Jun. 2016 11°06′56″ E Microstomum laurae 58°52′29″ N, MF185713 Saltö, Sweden 17 Jun. 2016 11°08′41″ E AJ405979** United Kingdom Lapptrasket, 60°03′07″ N, KP730567* 6 Aug. 2008 Microstomum lineare Finland 23°40′19″ E 61°39′35″ N, MF185697-9 Tensjön, Sweden 2 Jul. 2016 15°14′52″ E Koenigshafen, 55°02′24″ N, Microstomum papillosum KP730570* 8 Mar. 2007 Germany 8°23′52″ E Fiskebäckskil, 58°14′59″ N, Microstomum rubromaculatum MF185684-96 20 Aug. 2015 Sweden 11°26′45″ E Mangrove Bay, 25°52′15″ N, Microstomum sp. “B” KP730580* 11 Jan. 2009 Egypt 34°25′04″ E 42°34′29″ N, Microstomum sp. “D” KP730576* Pianosa, Italy 30 Apr. 2010 10°03′59″ E KP730562*, Sant Andrea 42°48′31″ N, KP730575*, 26–30 Apr. 2010 Bay, Italy 10°08′30″ E Myozonaria fissipara KP730577* 42°34′29″ N, KP730574* Pianosa, Italy 30 Apr. 2010 10°03′59″ E Fetovaia Bay, 42°43′36″ N, KP730573* 26 Apr. 2010 Italy 10°09′33″ E Myozonaria bistylifera Sant Andrea 42°48′31″ N, KP730584* 26 Apr. 2010 Bay, Italy 10°08′30″ E KP730569*, 42°34′29″ N, Myozonariinae Pianosa, Italy 30 Apr. 2010 KP730572* 10°03′59″ E and then back-translated to nucleotides. The general time-reversible model with gamma distribution and proportion invariant sites was determined to be the best model of sequence evolution with three substitution schemes by jModelTest2 (Tamura et al. 2011) based upon the Akaike information criterion (Akaike 1974). Maximum Likelihood Analysis (ML) was performed in RaxmlGUI v. 1.5b1 (Silvestro & Michalak 2012) with 1000 fast bootstrap replicates. Patristic distances were calculated using the TN93-model (Tamura & Nei 1993) with rate variation among sites and gamma distribution. Alignment gaps and ambiguous sites were not considered. 3 European Journal of Taxonomy 398: 1–18 (2018) Results Taxonomy Order Macrostomida Karling, 1940 Family Microstomidae Luther, 1907 Genus Microstomum Schmidt, 1848 Microstomum laurae sp. nov. urn:lsid:zoobank.org:act:49378EBE-7046-49D3-BC3C-DD783580A90E Fig. 1 Diagnosis Strap-shaped Microstomum with body length of 760 µm (two vegetative zooids) and rounded anterior and posterior ends. Posterior rim with approximately twenty adhesive papillae. Paired red eyespots 43 µm long and located in the lateral margins at level of the brain. Rhabdites concentrated in the anterior end above the pharynx. Nematocysts present. Preoral gut extending anterior to brain. Reproductive system unknown. GenBank accession number for partial COI sequence MF185712-3. Etymology This species is dedicated to Laura R. Atherton, mother of the first author. Material examined Holotype SWEDEN: vegetative specimen, Strömstad, Saltö, 58°52′29″ N, 11°08′41″ E, 10 cm, 17 Jun.
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